Rheumatoid arthritis is a systemic disease characterized by muscular pain and stiffness as well as articular inflammation and destruction. These symptoms are primarily the result of antibodies, sometimes referred to as autoantibodies, which react with an individual's own immunoglobulin antibodies. The precise cause of rheumatoid arthritis is unknown, but there is evidence that three different factors are involved, namely: (1) genetic predisposition to rheumatoid arthritis; (2) environmental factors, such as viral infection; and, (3) a functional defect in T-lymphocytes. The interrelationship among the genetic, environmental, and immunologic factors is not known.
Evidence of altered immune functions which bring about symptoms of rheumatoid arthritis include hypergammaglobulinemia, decreased in vivo and in vitro T-lymphocyte reactivity, and the presence of autoantibodies to immunoglobulin G (IgG). These "antiantibodies" or "antiimmunoglobulins" have been named rheumatoid factors (RF) because of their association with rheumatoid arthritis. RF has also been found with varying frequency in patients with most of the connective tissue diseases, many chronic and sub-acute infections, and a variety of miscellaneous disorders. In addition, RF has been found in many apparently healthy persons, particularly the elderly.
In spite of its lack of specificity for the diagnosis of rheumatoid arthritis, RF is still of value as a prognostic indicator. For example, studies have shown that high quantities or titers of RF are associated with destructive joint disease, the presence of rheumatoid nodules, and the likelihood of developing numerous systemic complications. Changes in the titer of RF during the course of the illness are not very helpful in assessing the course of the disease in a given individual. Nonetheless, in studies of groups of patients treated with pharmacologic agents, mean titers of RF generally decline if improvement occurs. Over one-half of patients with seropositive rheumatoid arthritis who go into remission become seronegative. However, about 25% of such individuals continue to have high titers in their sera in spite of clinical recovery.
The multivalent RF autoantibody specifically binds to the monovalent Fc portion, or tail, of the IgG class of immunoglobulin antibodies. RF is thus capable of binding to the IgG antibody while one or more of the binding arms of the IgG antibody is/are bound to its corresponding antigen. In fact, it is believed that RF is more attracted to the IgG antibody in its bound form due to conformational changes that take place in the IgG antibody, rendering the Fc portion of the antibody more accessible.
The most commonly used test for RF is the latex agglutination method of Singer and Plotz, sometimes referred to as the latex fixation test. Singer, J. M., and Plotz, C. M., "The latex fixation test. I. Application to the serologic diagnosis of rheumatoid arthritis", Am. J. Med., 21, 888-892 (1956). In this test, latex particles coated with human IgG are agglutinated on a slide, usually at a 1:20 dilution of test sample. The test sample is typically human serum, although Synovial fluid (taken from a suspected arthritic joint), is also analyzed on occasion. This test is generally regarded as a screening procedure from which a yes/no result can be obtained. A gross estimate of titer can be achieved by further serial dilutions of the serum utilizing the original latex tube test. In the latex tube test, the latex particle is agglutinated in solution, with the resulting agglutination being visually interpreted according to various standards and controls. The tube dilution test has the advantage of being considerably more sensitive than the slide test, but it is more cumbersome to perform.
The latex agglutination test, which is performd visually rather than instrumentally (such as with nephelometry), is the principle behind most of the subsequently devised methods for detecting RF. These methods all involve use of an indicator system such as latex, bentonite, or erythrocytes, to which human IgG is attached. The presence of RF is recognized agglutination, flocculation, or precipitation of the various indicator systems.
Heat-aggregated IgG may also be used directly as the antigen or indicator system in the assay. The heat aggregation step is believed by some to impart a conformational change to the IgG resulting in better detection. The conformational change is believed to simulate the conformational change encountered when the IgG is bound to its corresponding antigen through one or more of its binding arms.
An earlier test known as the sensitized sheep cell agglutination test (Waaler-Rose test) is still employed in some clinical laboratories. Sheep cells coated with rabbit antibody to these erythrocytes are agglutinated by certain RF. However, it is important first to remove any anti-sheep cell antibodies from the test sample by suitable absorption so as not to produce false-positive test reactions. Moreover, one must use fresh sheep cells that are standardized each day before use. This necessity for fresh cells makes the assay somewhat awkward to perform on a routine basis, although some believe the sheep cell agglutination test may have more specificity for the RF of rheumatoid arthritis.
Other available tests are a more direct variation of the original latex agglutination test. For example, a flocculation test using bentonite particles, rather than latex, coated with aggregated human IgG as the indicator system is used in some laboratories. Formalinized and "tanned" sheep cells; i.e., cells which have been preserved, can be coated with aggregated human IgG as the indicator system. These cells are then agglutinated by RF. This test is very sensitive, but the assay is somewhat more difficult to perform than the latex tests and may not lead to any practical advantage in routine studies. A radioimmunoassay for IgM RF has also been developed wherein insolubilized IgG is used as an immunoabsorbent from which RF can be eluted and characterized. Immunodiffusion tests have likewise been developed, employing both double and single diffusion methodologies.
Automated nephelometric or turbidimetric procedures can generally be employed using any variety of indicator systems. In turbidimetry, the reduction of light transmitted through the suspension of particles, or aggregates, is measured. The reduction is caused by reflection, scatter, and absorption of the light by the aggregates. In nephelometry, it is the light scattered or reflected toward a detector that is not in the direct path of light which is measured. Where automation is unavailable or impractical, a simpler version of nephelometry and turbidimetry is available wherein the amount of aggregation observed in a test sample is visually compared to a single standard or a series of standard controls.
In addition to the aforementioned indicator systems, heat aggregated IgG can be used directly as the indicator system. The primary drawback to the use of heat aggregated IgG as the indicator system is the uncontrolled, random orientation of the Fc portion of the IgG that is obtained in the heat aggregation process. Where latex, bentonite, or similar particles are used at the core of the indicator system, either intact IgG antibodies or Fc fragments can be used, with the number and orientation of Fc binding sites more easily controlled, depending upon the particular method of attachment employed. Regardless of the type of indicator system selected, nephelometric and turbidimetric methods provide a major advantage in that they are an objective analysis by an instrument of what has traditionally been a visual, highly subjective assessment of particle agglutination expressed in titration steps.
Serum or synovial fluid to be tested for RF must ordinarily be heated at 56.degree. C. before assay to inactivate the labile complement component Clq. The Clq component, frequently present in serum samples, has been described as a multivalent "bunch of tulips" wherein each "tulip" is capable of binding to the same Fc portion of the IgG antibody as RF. Consequently, any Clq present in a test sample is also capable of agglutinating particles coated with IgG, thus resulting in false-positive reactions.
The entire heat inactivation step consumes approximately 45 minutes. Each sample must first be subjected to incubation at 56.degree. C..+-.1.degree. C. for 30 minutes .+-.1 minute. The samples are then microcentrifuged for 5 minutes. Further sample handling uses the remaining approximately 10 minutes. This required pretreatment of samples imposes an additional time-consuming procedure in RF assay that must generally be incorporated into every test.
In some instances the pretreatment, or heat inactivation step, has been avoided where the latex slide agglutination and/or latex tube tests have been employed. Avoidance of the heat inactivation step in these tests has generally required the use of a standard glycine buffer and large dilutions of Clq. For example, serial dilutions of 1:20 through 1:10240 are used in the standard latex tube test. The glycine compound is believed to have some inhibitory effect on Clq.
Each RF assay, however, has different requirements with regard to characteristics such as assay precision. Most nephelometric and turbidimetric assays, for example, provide objective numerical data, as opposed to visual interpretation within a given concentration range. These automated types of nephelometric and turbidimetric assays are unable to tolerate the heavy dilutions of serum sample required in the visual latex agglutination and latex tube tests to sufficiently dilute out the interfering Clq from a test sample. Adjustment of this parameter is therefore unavailable in typical nephelometric and turbidimetric RF assays.
Use of a glycine buffer alone has proved to be ineffective in universal application to automated nephelometric and turbidimetric RF assays. Other known Clq inhibitors, such as diaminobutane or deoxyribonucleic acid (DNA) disclosed in U.S. Pat. No. 4,153,417, have likewise proved to be unsatisfactory in these types of nephelometric and turbidimetric assays. These compounds are either ineffective in inhibiting Clq activity or concurrently inhibit RF activity to the detriment of the assay.
Because of the expense of the latex tube test, in terms of glassware, reagents, and technician time, and because of the deficiencies of the slide agglutination test, automated nephelometric and turbidimetric tests have become highly desirable. It would be advantageous to provide such a test without the requirement of a separate pretreatment step of inactivate Clq.